Rho signaling participates in membrane fluidity homeostasis.

Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.

Abstract

Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeast's sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation.

All proteins shown have been implicated in the CWI pathway or in control of Rho1. All three known Rho1 GEFs (Rom1, Rom2, and Tus1) and all four known Rho1 GAPs (Lrg1, Bem2, Bag7, and Sac7) are shown. Underlining indicates proteins found in the screen. Proteins drawn in red are those which when mutated cause sensitivity to C16:1 but not C18:1. Sac7 is drawn in blue because sac7Δ instead causes C16:1R C18:1S.

(A) Three, ten-fold dilutions of yeast suspensions were plated on YPD+1% tergitol containing the indicated levels of free fatty acids. The five left-most strains were identified by the screen. The sixth, seventh, and eighth strains from the left were constructed by mating strains with single deletions, sporulating the diploids and dissecting tetrads. sap190Δ (C16:1R C18:1S) and fen1Δ (C16:1S C18:1S), both identified by the screen, are included as controls. Growth was at 30° for 3 days. (B) YPD+1% tergitol containing 1 M sorbitol. (C) pkc1Δ strains are acutely sensitive to C16:1. Asci from a sporulated PKC1/pkc1Δ diploid were dissected on 3 slabs of YPD medium containing 1 M sorbitol (YSD; 3 ml per microscope slide) and germinated at 30° for 20 hr. to give colonies containing between 10 and 200 cells. Agar slabs were then slid onto YSD plates containing C16:1 or C18:1 and grown at 30° for 4 days. This two-step procedure allowed attribution of colony size to the effect of fatty acids on vegetative growth rather than to an effect on spore germination. Spore viability on the 3 slabs ranged from 75 to 80%.

Growth properties of mutants are consistent with impaired regulation of membrane fluidity.

(A) The effects of C16:1 and C18:1 on growth counteract each other. Six strains were grown on YPD medium containing 1 M sorbitol and 1% tergitol supplemented with C16:1 (right panels) or not (left panels) and/or two levels of C18:1 (two bottom pairs of panels) or not (upper panels) and grown at 30° for 3 days. (B) Representation of the interplay between fatty acid composition, the proposed membrane fluidity homeostasis (MFH) signaling pathway, and temperature (T) on membrane fluidity. (C) C16:1-sensitivity of tus1Δ is suppressed by growth at 16°. Yeast were grown on YPD with or without C16:1 for 11 days (16°) or 2 days (30°). (D) The ts of tus1Δ is suppressed by C18:1. Growth on YPD with or without C18:1 was for 3 days. (E) The cs of sac7Δ is suppressed by either C16:1 or BA. YPD medium was supplemented with C16:1 or C18:1 or with benzyl alcohol (BA). (F) Growth of only one class of rho1ts strains is inhibited by C16:1 at permissive temperatures, and is enhanced by C18:1 at a semi-permissive temperature.

Total PL from each of four strains was purified in multiple experiments and acyl chain content quantified ( contains the complete data set). Mutations caused statistically significant changes in the molar ratios of (A) C16:1/C16:0 and of (B) C16:1/C18:1, but in neither the C18:1/C18:0 nor the C16:0/C18:0 ratios (). Error bars are the standard error of the mean. P values compared to wild type by paired, two-tailed Student's t-test are shown for tus1Δ and for bck1Δ ste11Δ ssk22Δ.

Losses of Rho1 GEF and GAP activities have opposing effects on PM fluidity.

Using TMA-DPH as a probe, fluorescence anisotropy was performed on wild type, erg6Δ, tus1Δ, sac7Δ, and bck1Δ ste11Δ ssk22Δ (“MK3Δ”), grown logarithmically in the absence (−) or presence of either 10 µM C18:1 or 10 µM C16:1. Addition of up to 100-fold more C18:1 or C16:1 had no additional effect. Anisotropy values are expressed relative to wild type (in the absence of either soap) and are shown as the mean ± S.E.M calculated from at least 3 independent experiments. *, p≤0.005 vs. wild type by paired, two-tailed Student's t-test.